title: Live imaging of transgenic mice expressing FRET biosensors.
abstract: In recent years, fluorescence imaging has received particular attention, due to increasing availabilities of fluorescent proteins and dyes, which had driven the development of novel biosensors. Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to difficulties in stable expression of FRET biosensors. In this study, we report efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were generated by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harboring Tol2 recombination sites. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kamioka
authors.forename: Kenta
authors.initials: K
authors.lastname: Sumiyama
authors.forename: Rei
authors.initials: R
authors.lastname: Mizuno
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Matsuda
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001565:Benzoates / Q000494:pharmacology; D015374:Biosensing Techniques; D003994:Bucladesine / Q000494:pharmacology; D002465:Cell Movement; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D007421:Intestine, Small / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D012867:Skin / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D013806:Theophylline / Q000494:pharmacology; D059008:Time-Lapse Imaging
chemical_list: D001549:Benzamides; D001565:Benzoates; D005456:Fluorescent Dyes; C094099:LY 293111; D003994:Bucladesine; C506614:mirdametinib; D004159:Diphenylamine; D013806:Theophylline; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases